Synthetic Spliceosome Probes and Modulators

The Stamm lab will test the effect of compound 15 and other spliceostatin derivatives on alternative and constitutive splicing. For the work on alternative splicing, we will test the drug on at least 20 reporter genes that we know are regulated by PPI ((1) and our unpublished data). We will also clone newly discovered compound-I 5 regulated alternative exons into a splicing reporter system that we developed (2) and test the resulting constructs in in vivo assays. The effect of compound 15 on splicing will be tested in vitro using a phosphorylation assays. HeLa nuclear extract will be incubated with 32P-gamrna ATP in the presence/absence of the compound and a change in phosphorylation will be detected by autoradiography followed by mass-spectrometry. Identified proteins will be validated using immunoprecipitation and the effect of the phosphorylation will be assessed by generating serine to arginine and serine to glutamic acid mutated proteins. The mutant proteins will be tested in splicing assays both in vivo and in vitro.